A molecular FRT (Flp recombinase recognition target)-based cassette system for multiple gene disruption in the yeast Saccharomyces cerevisiae was developed. FRT DNA sequences were designed with different core mutations and subsequently cloned in direct orientation upstream and downstream of a marker gene to serve as template for the amplification of a set of different gene disruption cassettes. After each disruption, the marker can be easily eliminated from its integration site by in vivo site-specific recombination between the two identical, mutated FRT sequences flanking the marker, leaving behind one FRT sequence with a particular point mutation. Since recombination between two FRTs with a different core mutation is extremely rare, the possibility of chromosome rearrangements, due to site-specific recombination between residual FRTs, is very low. In strains containing 2-m ([cir + ]) the site-specific reaction is catalysed by the endogenous Flp gene product, whereas in strains without 2-m ([cir 0 ]), the FLP gene is carried on the cassette, together with the marker gene. This system can be applied for haploid and diploid [cir + ] and [cir 0 ] strains.
When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.
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